Immunoglobulin-binding polypeptides

ABSTRACT

A method of separating V H  containing immunoglobulins by admixing an immunoglobulin binding peptide is described. The polypeptide of the invention is capable of binding to heavy chain variable regions of antibodies in an antigen independent manner and can separate antigen/antibody complexes.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part application of copending application Ser.No. 07/404,968, now allowed U.S. Pat. No. 5,231,167, having the sametitle and filed Sep. 8, 1989, the disclosure of which is herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to a new class of immunoglobulin-bindingpolypeptides, more particularly, the present invention relates tosynthetic polypeptides and chimeric proteins capable of binding animmunoglobulin (Ig) molecule independent of the Ig molecule's antigenspecificity.

BACKGROUND

The CD4 receptor is a non-polymorphic glycoprotein having a molecularweight of about 60,000 daltons that is present primarily on the surfaceof T lymphocytes with helper/inducer function. Structurally, it consistsof three segments: extracellular, transmembrane and cytoplasmic. Theformer comprises four immunoglobulin (Ig) variable (V) region-likedomains of which the first, second and forth are linked by disulfidebonds. Because of sequence and structure homology with the V domain ofIg light (L) chain, it has been proposed that the gene coding for theCD4 molecule is a member of the Ig supergene family.

While it is clear that CD4 is a receptor that binds biologicallyimportant molecules, its physiological role is not fully understood.

Although it was long suspected that CD4 may function as a receptormolecule for MHC class II antigens, only recently was it demonstratedthat fibroblasts transfected with a CDNA coding for CD4, and expressinghigh levels of this protein, bound tightly to human B cells bearingMHC-coded class II molecules. See for example, Guy et al., Nature,328:626-629 (1987); Doyle et al., Nature, 330:258-259 (1987); andSleckman et al., Nature, 328:351-353 (1987). This CD4-MHC Class IIinteraction was inhibited by either anti-CD4 or anti-MHC class IIantibodies.

Several studies have provided evidence that the CD4 molecule acts as areceptor for the human immunodeficiency virus type 1 (HIV-1). Forinstance, Jameson et al., Science., 240:1335-1338 (1988) reported thatthe binding site for HIV-1 on CD4 is probably formed by amino acidresidues 16-49.

Other receptors of interest to the present invention are those whichbind the constant (Fc) portion of immunoglobulin molecules. Forinstance, bacteria of several gram-positive species produce proteinswhich bind to the Fc region of Ig. For example see the best known ofthese Fc receptors is protein A of Staphylococcus aureus. Protein A hasbeen widely used in laboratory and clinical diagnostic immunochemicalprocedures which exploit the ability to bind to a variety ofimmunoglobulin G (IgG) antibodies independently of antigen association.

The gene coding for protein A has been cloned, its CDNA sequencedetermined and its amino acid residue sequence deduced. See LoFdahl etal., Proc. Natl. Acad. Sci. USA, 80:697-701 (1983); Uhlen et al., J.Biol. Chem., 259:1695-1702 (1984); and Moks et al., Eur. J. Biochem.,156:637-643 (1986). In addition, those portions of protein A responsiblefor IgG-binding have been identified by Moks et al., Eur. J. Biochem.,156:637-643 (1986), as consisting of five highly homologous segments,designated A-E, having sizes ranging from 50 (segment E) to 61 (segmentD) amino acid residues.

Fc receptors with broader specificity than protein A are produced byStreptococcus species, especially those of Lancefield groups C and G.See for example, Bjorck et al., J. Immunol., 133:969-974 (1984);Langone, I. I., Adv. Immunol., 32:157-252 (1982); Myhre et al., Infect.Immunol., 17:475-482 (1977); and Reis et al., Mol. Immunol., 23:425-431(1986). The protein produced by group G Streptococcus species known asprotein G, has been shown to bind to all four classes of human IgG,including IgG3, to which protein A does not bind by Bjorck et al., J.Immunol., 133:969-974 (1984). Furthermore, protein G binds more stronglythan protein A to several animal IgG classes and mouse monoclonalantibodies as demonstrated by Akerstron et al., J. Immunol.,135:2589-2592 (1985).

The gene for protein G has been cloned, its cDNA sequence determined andits amino acid residue sequence deduced. See Fahnestock et al., J.Bact., 167:870-880 (1986). Like protein A, the structure of theIgG-binding regions of protein G have been determined by Guss et al.,EMBO J., 5:1567-1575 (1986). Those regions, designated C1, C2 and C3,each contain 55 amino acid residues and are separated within the proteinby two "spacers", designated D1 and D2 of 16 amino acid residues each.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that the CD4 receptor binds theimmunoglobulin heavy chain variable region (V_(H)) in a non-immunemanner, i.e., in an antigen-independent manner.

The present invention is based on the elucidation of theantigen-independent V_(H) -binding sites on the CD4 protein and thedetermination of the amino acid residue sequences of those portions ofthe CD4 protein to which _(v) H molecules bind. It is believed that atleast three V_(H) -binding sites exist on the CD4 molecule, one locatedin the region of CD4 amino acid residues 30 to 40, another in the regionformed by CD4 amino acid residues 60 to 90, and a third in the region ofCD4 amino acid residues 130 to 160. Within the region of CD4 defined byresidues 30-40, the sequence of amino acid residues represented by theformula:

    -Ile-Lys-Ile-Leu-

(CD4 residues 34 to 37) appears to play a particularly important role inantigen-independent Ig-binding. Furthermore, the results presentedherein indicate that the region formed by CD4 amino acid 24 to 29 playsan important role in increasing the affinity of CD4 residues 34 to 37for V_(H).

Knowledge of these amino acid residue sequences has enabled thesynthesis of polypeptides of the present invention which are capable ofbinding V_(H) molecules, thereby allowing for their location and/orseparation. As is readily apparent, these polypeptides can be used toisolate V_(H) molecules from complex mixtures of proteins or forremoving all or a portion of the V_(H) molecules from a complex mixtureof proteins. The subject polypeptides bind V_(H) molecules when theV_(H) molecules are present in the immunologically bound as well as freeforms. That is, the subject polypeptides bind to immune complexes andcan be used to identify the presence of such complexes or to isolatethose complexes.

In addition, it has also been discovered that operatively linking aV_(H) -binding polypeptide of the present invention to a V_(H) moleculeincreases the affinity of the V_(H) molecule for its antigen, or, in thecase of a catalytic V_(H) molecule, the V_(H) molecule substrateturn-over rate.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of the specification:

FIGS. 1A-1 and 1A-2 illustrate the amino acid residue sequence of theCD4 molecule and a nucleotide sequence coding for it. The sequences arethose described by Maddon et al., Cell, 42:93-104 (1985), as modified byLittman et al., Cell, 55:591 (1988). See Bialy, Biotech., 6:1268 (1988)for a further explanation of the modification and the reasons therefor.

FIGS. 2A-1 and 2A-2 illustrate a gene coding for a StaphylococcalProtein A and its deduced amino acid residue sequence as discussed byUhlen et al., J. Biol. Chem., 259:1695-1702 (1984). FIG. 2B illustratesthe amino acid residue sequences of preferred Protein A F_(C) -bindingsegments and nucleotide sequences coding for those segments. E, D, A, Band C are the different F_(C) -binding domains of staphyloccal ProteinA; Z is the synthetic F_(C) -binding domain described in Nilsson et al.,Protein Eng., 1:107-113 (1987). Amino acid differences are shown. Anasterisk (*) represents a silent mutation, i.e. a change in the DNAsequence that does not alter the amino acid sequence coded for. A hyphen(-) represents the same DNA codon that is present in the Z domain. Theunderlined amino acids in the Z sequence are involved in the binding toFc as described in Moks et al., Eur. J. Biochem., 156:637-643 (1986).The amino acids forming two x-helices are boxed.

FIGS. 3A-1 and 3A-2 illustrate a gene coding for a streptococcal ProteinG and its deduced amino acid residue sequence as described by Guss etal., EMBO J., 5:1567-1575 (1986). FIG. 3B illustrates the base sequencesand amino acid residue sequences encoded thereby of preferred Protein GF_(C) -binding segments. The identical DNA codons are indicated with ahyphen (-) and silent DNA codon changes are indicated with an asterisk(*).

In FIG. 4, antibodies not immunospecific for peptide p21-49 wereexamined at two concentrations (40 ug/ml and 4 ug/ml) for their abilityto be bound by p2l-49 in an antigen-independent manner. In each panel,the amount of binding is indicated by the optical density, whichincreases with greater binding. FIG. 4A illustrates the binding of V_(H)-binding peptide p21-49 to 22 murine monoclonal antibodies of differentantigen specificities and isotypes (groups I, II and III). FIG. 4Billustrates the binding of V_(H) -binding peptide p21-49 to 47 humanmyeloma proteins of different isotypes (groups I-VIII).

In FIG. 5, the inhibition of monoclonal antibody binding to solid-phasepeptide p21-49 by liquid-phase (soluble) peptide p21-49 is shown. Inaddition, the apparent binding constants of each monoclonal antibody,determined according to the method of Nieto et al., Mol.Immunol., 21:537(1984).

FIG. 6A illustrates the enhancement of antibody binding to V_(H)-binding peptide p21-49, by the binding of antigen to antibody asevidenced by increasing optical density with in increasing antigen (Tg)concentration. FIG. 6B illustrates peptide p21-49 does not inhibit thebinding of antibody to antigen (antithyroglobulin to thyroglobulin) asevidenced by the approminately equivalent optical density observed ateach peptide concentration.

In FIG. 7, the inhibition of V_(H) -binding peptide p21-49 binding toimmunoglobulin in the presence of various concentrations of dextransulfate is illustrated. The monoclonal antibodies used were 62,immunospecific for thyroglobulin and OKT4A, immunospecific for nativeCD4.

FIG. 8A illustrates the binding of V_(H) -binding peptide p21-49 tointact antibody, isolated Ig heavy chain (H chain) and isolated Ig lightchain (L chain). FIG. 8B illustrates the binding of V_(H) -bindingpeptide p21-49 to chimeric (mouse/human) antibodies of the IgG, (human)isotype with different isotypes of light chain (kappa and lambda). FIG.8C illustrates the binding of V_(H) -binding peptide p21-49 to intactantibody, F(ab')₂ fragments and Fab fragments. FIG. 8D illustrates thebinding of V_(H) -binding peptide p21-49 to mutant antibodies containingdeletions in either the CH3 domain or the CH1 domain of theimmunoglobulin heavy chain.

In FIG. 9, the commercially available pRIT5 Protein A gene fusion vectoris shown. The plasmid pRIT5 is designed to permit high-level expressionof fusion proteins containing the staphylococcal Protein A F_(C)-binding region in both E. coli and S. aureus host cells as described inNilsson et al., EMBO J., 4:1075 (1985). Genes inserted into the multiplecloning site are expressed from the Protein A promoter and translocatedto the periplasmic space in E. coli, or secreted into the growth mediumin gram-positive cells. The Protein A promoter and signal sequence arefunctional in a wide range of bacterial species including both E. coliand S. aureus as described by Uhlen et al., J. Bact., 159:513 (1984).The plasmid also contains the broad-host-range origin of replication andchloramphenicol acetyltransferase gene from pCl94 as described inHorinouchi et al., J. Bact., 150:815 (1982).

In FIG. 10, a preferred double stranded synthetic DNA sequence (gene)coding for V_(H) -binding peptide p21-49 is illustrated. The sequencecoding for V_(H) -binding peptide p21-49 is flanked by Bam HIrestriction endonuclease sites to allow this sequence to be easilyinserted into the pRIT5 vector.

In FIG. 11, the pASI-3 expression vectors containing the staphylococcalProtein A promoter and signal sequence described in Abrahmsen et al.,EMBO J., 4:3901-3906 (1985) is shown. The pASI-3 vector series isdesignated to permit high level expression of a gene inserted into themultiple cloning site in both E. coli and S. aureus host cells. Boxesrepresent the position of the genes coding for the staphylococcalProtein A signal sequence (s), the B-bactamase (Amp) and chloramphenicolacetyl transferase (cml). The origins of replication in E. coli (oriEC)and in staphylococci/bacilli (oriBS) are also shown.

In FIG. 12, a preferred synthetic DNA sequence (gene) coding for theF_(C) -binding portion of streptococcal Protein G is shown. Thesynthetic DNA gene coding for the Protein G F_(C) -binding region isflanked by Eco RI restriction endonuclease sites to allow this sequenceto be easily inserted into the PASI vector.

In FIG. 13, a preferred synthetic double stranded DNA sequence (gene)coding for the streptococcal Protein G F_(C) -binding region is shown.The synthetic gene coding for the Protein G F_(C) -binding region isflanked by DNA sequences compatible with Bam HI restriction endonucleasesites.

FIG. 14 illustrates the affinity purification of a monoclonal antibody(IgG₁,k) from a complex mixture of proteins (ascites fluid) using ap21-49-Sepharose 4B column. The total protein (solid circles) and IgG₁,kportion of each eluted fraction are shown. The arrows indicate when thesample and elution buffer were applied to the column.

FIG. 15 reports the binding of various synthetic CD4 peptides spawningfrom amino acid residue 21 to 38 of the first extracellular domain ofCD4. The binding of recombinant CD4 (rCD4) and p21-29 are shown aspositive controls.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: The amino acid residues described herein arepreferred to be in the "L" isomeric form. However, residues in the "D"isomeric form can be substituted for any L-amino acid residue, as longas the desired functional property of immunoglobulin-binding is retainedby the polypeptide. NH2 refers to the free amino group present at theamino terminus of a polypeptide. COOH refers to the free carboxy grouppresent at the carboxy terminus of a polypeptide. A hyphen at the amino-or carboxy-terminus of a sequence indicates the presence of a furthersequence of amino acid residues or a respective NH₂ or COOH terminalgroup. In keeping with standard polypeptide nomenclature, J. Biol.Chem., 243:3552-59 (1969), abbreviations for amino acid residues areshown in the following Table of Correspondence:

    ______________________________________                                        TABLE OF CORRESPONDENCE                                                       SYMBOL                                                                        1-Letter  3-Letter       AMINO ACID                                           ______________________________________                                        Y         Tyr            tyrosine                                             G         Gly            glycine                                              F         Phe            phenylalanine                                        M         Met            methionine                                           A         Ala            alanine                                              S         Ser            serine                                               I         Ile            isoleucine                                           L         Leu            leucine                                              T         Thr            threonine                                            V         Val            valine                                               P         Pro            proline                                              K         Lys            lysine                                               H         His            histidine                                            Q         Gln            glutamine                                            E         Glu            glutamic acid                                        W         Trp            tryptophan                                           R         Arg            arginine                                             D         Asp            aspartic acid                                        N         Asn            asparagine                                           C         Cys            cysteine                                             ______________________________________                                    

It should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.

Polypeptide: refers to a linear series of amino acid residues connectedto one another by peptide bonds between the alpha-amino groups andcarboxy groups of contiguous amino acid residues.

Peptide: as used herein refers to a linear series of not more than about50 amino acid residues connected one to the other as in a polypeptide.

Protein: refers to a linear series of greater than 50 amino acidresidues connected one to the other as in a peptide.

Synthetic peptide: refers to a chemically produced chain of amino acidresidues linked together by peptide bonds that is free of naturallyoccurring proteins and fragments thereof.

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1' carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3' or 5'position of the pentose it is referred to as a nucleotide. A sequence ofoperatively linked nucleosides is typically referred to herein as a"nucleotide sequence", and is represented herein by a formula whose leftto right orientation is in the conventional direction of 5' terminus to3' terminus.

Immunoglobulin: refers to intact immunoglobulin molecules andimmunologically active portions (capable of binding antigens) ofimmunoglobulin molecules. Exemplary imunoglobulin molecules are thoseportions of intact antibody molecules known in the art as V_(H), Fab,Fab¹, F(ab¹)₂ and F(v).

B. Polypeptides

In one embodiment, a V_(H) -binding polypeptide of the present inventionhas an amino acid residue sequence corresponding to a portion of thesequence of the CD4 molecule, exhibits antigen-independent affinity forthe V_(H) region of Ig molecules, and is substantially, free from theability to bind the human immunodeficiency virus, e.g., HIV-1 and/orHIV-2. Methods for determining the ability of a CD4-related polypeptideto bind HIV are well known in the art and typically involve examiningthe ability of a polypeptide to inhibit HIV-induced cell fusion(syncytium formation). See, for example, Jameson, et al., Science,240:1335-1338 (1988). (The references cited herein are herebyincorporated by reference.)

Preferred polypeptides are those having an amino acid residue sequencecorresponding, and preferably identical, to a sequence shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Pep-                                                                          tide Amino Acid Residue Sequence                                              ______________________________________                                        p 16-                                                                              NH.sub.2 --Cys--Thr--Ala--Ser--Gln--Lys--Lys--Ser--Ile--                 38   Gln--Phe--His--Trp--Lys--Asn--Ser--Asn--Gln--Ile--                            Lys--Ile--Leu--Gly--COOH                                                 p 21-                                                                              NH.sub.2 --Lys--Lys--Ser--Ile--Gln--Phe--His--Trp--Lys--                 38   Asn--Ser--Asn--Gln--Ile--Lys--Ile--Leu--Gly--COOH                        p 22-                                                                              NH.sub.2 --Lys--Ser--Ile--Gln--Phe--His--Trp--Lys--                      38   Asn--Ser--Asn--Gln--Ile--Lys--Ile--Leu--Gly--COOH                        p 23-                                                                              NH.sub.2 --Ser--Ile--Gln--Phe--His--Trp--Lys--                           38   Asn--Ser--Asn--Gln--Ile--Lys--Ile--Leu--Gly--COOH                        p 24-                                                                              NH--Ile--Gln--Phe--His--Trp--Lys--                                       38   Asn--Ser--Asn--Gln--Ile--Lys--Ile--Leu--Gly--COOH                        p 21-                                                                              NH.sub.2 --Lys--Lys--Ser--Ile--Gln--Phe--His--Trp--Lys--                 37   Asn--Ser--Asn--Gln--Ile--Lys--Ile--Leu--COOH                             ______________________________________                                    

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is capable of binding Ig in anantigen-independent manner, i.e., as a nonantigenic ligand. Therefore, apresent polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in their use.

The term "analog" refers to any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinconservatively substituted with a functionally similar residue. Examplesof conservative substitutions include the substitution of one nonpolar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another, the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, between glycine and serine, the substitution of one basicresidue such as lysine, arginine or histidine for another, or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

The phrase "conservative substitution" also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

"Chemical derivative" refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-imbenzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite bindingactivity is maintained.

The term "fragment" refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

A subject polypeptide can be prepared using the solid-phase synthetictechnique initially described by Merrifield, in J. Am. Chem. Soc.85:2149-2154 (1963). Other polypeptide synthesis techniques may befound, for example, in M. Bodanszky et al., Peptide Synthesis, JohnWiley & Sons, 2d Ed., (1976) as well as in other reference works knownto those skilled in the art. A summary of polypeptide synthesistechniques may be found in J. Stuart and J. D. Young, Solid PhasePeptide Synthesis, Pierce Chemical Company, Rockford, Ill., 3d Ed.,Neurath, H. et al., Eds., p. 104-237, Academic Press, New York, N.Y.(1976). Appropriate protective groups for use in such syntheses will befound in the above texts as well as in J. F. W. McOmie, ProtectiveGroups in organic chemistry, Plenum Press, New York, N.Y. (1973).

In general, those synthetic methods comprise the sequential addition ofone or more amino acid residues or suitably protected amino acidresidues to a growing polypeptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amid linkage with the residue already attached to the solid support.The protecting group of the amino or carboxyl group is then removed fromthis newly added amino acid residue, and the next amino acid (suitablyprotected) is then added, and so forth. After all the desired aminoacids have been linked in the proper sequence any remaining terminal andside group protecting groups (and solid support) are removedsequentially or concurrently, to provide the final polypeptide.

The polypeptides of the present invention generally contain a V_(H)-binding segment of at least 10 amino acid residues and up to fiftyamino acid residues, preferably 10-35 amino acid residues. Thepolypeptides can be linked to an additional sequence of amino acids ateither or both the N-terminus and C-terminus, wherein the additionalsequences are from 1-100 amino acids in length. Such additional aminoacid sequences, or linker sequences, are heterologous to the CD4 aminoacid residue sequence and can be conveniently affixed to a detectablelabel, solid matrix, or carrier. Labels, solid matrices and carriersthat can be used with peptides of the present invention are describedbelow. Typical amino acid residues used for linking are tyrosine,cysteine, lysine, glutamic acid and aspartic acid, or the like.

Any polypeptide of the present invention, including a chimericpolypeptide as described hereinbelow, may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the polypeptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

The present invention further includes a composition that includes asubject polypeptide in combination with one or more of a pH bufferingagent, wetting agent, anti-oxidant, reducing agent, aqueous medium, andthe like, such composition being formulated as an aqueous solution for ause as described herein or as a dry composition, such as a powder, thatcan be reconstituted to form an aqueous solution.

C. Chimeric Polypeptides

Another V_(H) -binding polypeptide of this invention is a chimericpolypeptide comprising at least one first segment and at least onesecond segment operatively linked by a peptide bond to form a single(unitary) polypeptide chain. The first segment is a V_(H) -bindingsegment having an amino acid residue sequence corresponding to all or aV_(H) -binding portion of a CD4 molecule. Preferred V_(H) -bindingsegments have an amino acid residue sequence corresponding, andpreferably identical to, a sequence shown in FIG. 1 from about residue30 to about residue 40, from about residue 21 to about residue 26, fromabout residue 35 to about residue 38, from about residue 21 to aboutresidue 49, from about residue 16 to about residue 49, from aboutresidue 16 to about residue 38, from about residue 21 to about residue38, from about residue 22 to about residue 38, from about residue 21 toabout 38, from about residue 23 to about residue 38, from about residue24 to about residue 38, from about residue 29 to about residue 43, fromabout residue 32 to about residue 54, from about residue 38 to aboutresidue 62, from about residue 58 to about residue 82, from aboutresidue 66 to about residue 90, and from about residue 128 to aboutresidue 161.

Also preferred are chimeric polypeptides having one or more V_(H)-binding segments of about 50 residues or less, typically about 15residues to about 35 residues, wherein said segments have an amino acidresidue sequence that corresponds, and is preferably identical, to aV_(H) -binding portion of CD4, and wherein each V_(H) -binding segmentincludes the amino acid residue sequence -Lys-Lys-Ser-Ile-Gln-Phe-and/or -Lys-Ileu-Leu-Gly-.

Preferably, the second segment is at least about 5, preferably at leastabout 15 and more preferably at least about 25, amino acid residueshaving a sequence that is heterologous to the sequence of CD4. Aheterologous CD4 amino acid residue sequence is a sequence that does notimmunologically cross-react with CD4. In preferred embodiments, thesecond segment of a subject chimeric polypeptide is comprised of asequence of amino acid residues capable of binding the F, portion of anIg molecule, i.e. , is an F_(C) -binding segment.

The F_(C) -binding segment of a subject chimeric polypeptide has anamino acid residue sequence corresponding to all or a F_(C) -bindingportion of an F_(C) -binding protein. F_(C) -binding proteins are wellknown in the art. See, for example, Kronvall, J. Immunol., 111:1401-1406(1973); Shea, et al., Infect. Immunol., 34:851-855 (1981); Langone, Adv.Immunol., 32:157-252 (1982); Reis, et al., J. Immunol., 132:3091 (1984);Reis, et al., J. Immunol., 132:3098-3102 (1984); Bjorck, et al., J.Immunol., 133:969-974 (1984); Akerstrom, et al., J. Immunol.,135:2589-2592 (1985); and Myhre, et al., Infect Immun., 17:475-482(1977). Methods for cloning the genes coding for the proteins describedin the above articles, sequencing those genes and deducing the aminoacid residue sequence of the entire protein as well as the Ig-bindingsegments thereof, are well known in the art as discussed hereinbelow.

Particularly preferred F_(C) -binding proteins, all or a F_(C) -bindingportion of which can be used to form a subject chimeric polypeptide, arethose known in the art as Proteins A, Protein G and Protein Z. The aminoacid residue sequences of Proteins A and Z, as well as DNA sequencescoding for those proteins, are described in Lofdahl, et al., Proc. Natl.Acad. Sci. USA., 80:697-701 (1983) and Nilsson, et al., Prot. Eng.,1:107113 (1987). The amino acid residue sequence of Protein G, as wellas a DNA sequence coding for that protein, are described in Moks, etal., Eur. J. Biochem., 156:637-643 (1986); Guss et al., EMBO,5:1567-1575 (1986); Gahnestock, et al., J. Bact., 167:870-880 (1986);and Bjorck, et al., Mol. Immunol., 24:1113-1122 (1987).

Particularly preferred F_(C) -binding segments useful in forming asubject chimeric polypeptide have an amino acid residue sequencecorresponding, and more preferably identical, to at least a 10 aminoacid residue portion of a sequence shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Peptide                                                                            Amino Acid Residue Sequence                                              __________________________________________________________________________    (A).sup.1                                                                          --Thr--Tyr--Lys--Leu--Ile--Leu--Asn--Gly--Lys--Thr--Leu--                     Lys--Gly--Glu--Thr--Thr--Thr--Glu--Ala--Val--Asp--Ala--                       Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--Ala--                       Asn--Asp--Asn--Gly--Val--Asp--Gly--Glu--Trp--Thr--Tyr--                       Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--                       Lys--Pro--Glu--Val--Ile--Asp--,                                          (B).sup.1                                                                          --Thr--Tyr--Lys--Leu--Ile--Leu--Asn--Gly--Lys--Thr--Leu--                     Lys--Gly--Glu--Thr--Thr--Thr--Glu--Ala--Val--Asp--Ala--                       Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--Ala--                       Asn--Asp--Asn--Gly--Val--Asp--Gly--Glu--Trp--Thr--Tyr--                       Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--,                 (C).sup.2                                                                          --Thr--Tyr--Lys--Leu--Val--Ile--Asn--Gly--Lys--Thr--Leu--                     Lys--Gly--Glu--Thr--Thr--Thr--Glu--Ala--Val--Asp--Ala--                       Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--Ala--                       Asn--Asp--Asn-- Gly--Val--Asp--Gly--Glu--Trp--Thr--Tyr--                      Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--                       Lys--Pro--Glu--Val--Ile--Asp--,                                          (D).sup.2                                                                          --Thr--Tyr--Lys--Leu--Val--Ile--Asn--Gly--Lys--Thr--Leu--                     Lys--Gly--GLu--Thr--Thr--Thr--Glu--Ala--Val--Asp--Ala--                       Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--Ala--                       Asn--Asp--Asn--Gly--Val--Asp--Gly--Glu--Trp--Thr--Tyr--                       Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--,                 (E).sup.3                                                                          --Thr--Lys--Leu--Val--Ile--Asn--Gly--Lys--Thr--Leu--Lys--                     Gly--Glu--Thr--Thr--Thr--Lys--Ala--Val--Asp--Ala--Glu--                       Thr--Ala--Glu--Lys--Ala--Phe--Lys--Gln--Tyr--Ala--Asn--                       Asp--Asn--Gly--Val--Asp--Gly--Val--Trp--Thr--Tyr--Asp--                       Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--Lys--                       Pro--Glu--Val--Ile--Asp--,                                               (F).sup.3                                                                          --Thr--Try--Lys--Leu--Val--Ile--Asn--Gly--Lys--Thr--Leu--                     Lys--Gly--Glu--Thr--Thr--Thr--Lys--Ala--Val--Asp--Ala--                       Glu--Thr--Ala--Glu--Lys--Ala--Phe--Lys--Gln--Tyr--Ala--                       Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--Glu--,                 (G).sup.4                                                                          --Leu--Lys--Gly--Glu--Thr--Thr--Thr--Glu--Ala--Val--Asp--                     Ala--Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--                       Ala--Asn--Asp--Asn--Gly--Val--Asp--Gly--Glu--Trp--Thr--                       Tyr--Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--                       Glu--Lys--Pro--Glu--Val--Ile--Asp--,                                     (H).sup.4                                                                          --Leu--Lys--Gly--Glu--Thr--Thr--Thr--Glu--Ala--Val--Asp--                     Ala--Ala--Thr--Ala--Glu--Lys--Val--Phe--Lys--Gln--Tyr--                       Ala--Asn--Asp--Asn--Gly--Val--Asp--Gly--Glu--Trp--Thr--                       Tyr--Asp--Asp--Ala--Thr--Lys--Thr--Phe--Thr--Val--Thr--                       Glu--,                                                                   (I).sup.5                                                                          --Val--Asp--Asn--Lys--Phe--Asn--Lys--Glu--Gln--Gln--Asn--                     Ala--Phe--Tyr--Glu--Ile--Leu--His--Leu--Pro--Asn--Leu--                       Asn--Glu--Glu--Gln--Arg--Asn--Ala--Phe--Ile--Gln--Ser--                       Leu--Lys--Asp--Asp--Pro--Ser--Gln--Ser--Ala--Asn--Leu--                       Leu--Ala--Glu--Ala--Lys--Lys--Leu--Asn--Asp--Ala--Gln--                       Ala--Pro--Lys--,                                                         (J).sup.6                                                                          --Ala--Gln--His--Asp--Glu--Ala--Gln--Gln--Asn--Ala--Phe--                     Tyr--Gln--Val--Leu--Asn--Met--Pro--Asn--Leu--Ala--Asp--                       Glu--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--Leu--Lys--                       Asp--Asp--Pro--Ser--Gln--Ser--Ala--Asn--Val--Leu--Gly--                       Glu--Ala--Gln--Lys--Leu--Asn--Asp--Ser--Gln--Ala--Pro--                       Lys--,                                                                   (K).sup.7                                                                          --Ala--Asp--Asn--Asn--Phe--Asn--Lys--Asp--Gln--Gln--Ser--                     Ala--Phe--Tyr--Glu--Ile--Leu--Asn--Met--Pro--Asn--Leu--                       Asn--Glu--Ala--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--                       Leu--Lys--Asp--Asp--Pro--Ser--Gln--Ser--Thr--Asn--Val--                       Leu--Gly--Glu--Ala--Lys--Lys--Leu--Asn--Glu--Ser--Gln--                       Ala--Pro--Lys--,                                                         (L).sup.8                                                                          --Ala--Asp--Asn--Asn--Phe--Asn--Lys--Glu--Gln--Gln--Asn--                     Ala--Phe--Tyr--Glu--Ile--Leu--Asn--Met--Pro--Asn--Leu--                       Asn--Glu--Glu--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--                       --Ala--Asp--Asn--Asn--Phe--Asn--Lys--Glu--Gln--Gln--Asn--                     Ala--Phe--Tyr--Glu--Ile--Leu--Asn--Met--Pro--Asn--Leu--                       Asn--Glu--Glu--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--                       Leu--Lys--Asp--Asp--Pro--Ser--Gln--Ser--Ala--Asn--Leu--                       Leu--Ser--Glu--Ala--Lys--Lys--Leu--Asn--Glu--Ser--Gln--                       Ala--Pro--Lys--,                                                         (M).sup.9                                                                          --Ala--Asp--Asn--Lys--Phe--Asn--Lys--Glu--Gln--Gln--Asn--                     Ala--Phe--Tyr--Glu--Ile--Leu--His--Leu--Pro--Asn--Leu--                       Asn--Glu--Glu--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--                       Leu--Lys--Asp--Asp--Pro--Ser--Gln--Ser--Ala--Asn--Leu--                       Leu--Ala--Glu--Ala--Lys--Lys--Leu--Asn--Asp--Ala--Gln--                       Ala--Pro--Lys--, and                                                     (N).sup.10                                                                         --Ala--Asp--Asn--Lys--Phe--Asn--Lys--Glu--Gln--Gln--Asn--                     Ala--Phe--Tyr--Glu--Ile--Leu--His--Leu--Pro--Asn--Leu--                       Thr--Glu--Glu--Gln--Arg--Asn--Gly--Phe--Ile--Gln--Ser--                       Leu--Lys--Asp--Asp--Pro--Ser--Val--Ser--Lys--Glu--Ile--                       Leu-- Ala--Glu--Ala--Lys--Lys--Leu--Asn--Asp--Ala--Gln--                      Ala--Pro--Lys--,                                                         __________________________________________________________________________     .sup.1 The amino acid sequence of Streptococcal Protein G region C1           described in Guss, et al., EMBO J., 5:1567-1575 (1986).                       .sup.2 The amino acid sequence of Streptococcal Protein G region C2           described in Guss, et al., EMBO J.. 5:1567-1575 (1986).                       .sup.3 The amino acid sequence of Streptococcal Protein G region C3           described in Guss, et al., EMBO J., 5:1567-1575 (1986).                       .sup.4 The amino acid sequence coded by the Cla I restriction enzyme          fragment described in FIG. 4 of Guss, et al., EMBO J.. 5:1567-1575 (1986)     .sup.5 The amino acid sequence of the Z protein described in Nilsson, et      al., Protein Eng., 1:107-113 (1987).                                          .sup.6 The amino acid sequence of the E domain of Staphylococcal Protein      described in Nilsson, et al., Protein Eng., 1:107-113 (1987).                 .sup.7 The amino acid sequence of the D domain of Staphylococcal Protein      described in Nilsson, et al., Protein Eng., 1:107-113 (1987).                 .sup.8 The amino acid sequence of the A domain of Staphylococcal Protein      described in Nilsson, et al., Protein Eng., 1:107-113 (1987).                 .sup.9 The amino acid sequence of the B domain of Staphylococcal Protein      described in Nilsson, et al., Protein Eng., 1:107-113 (1987).                 .sup.10 The amino acid sequence of the C domain of Staphylococcal Protein     A described in Nilsson, et al., Protein Eng., 1:107-113 (1987).          

The antigen-dependent Ig-binding segments (i.e., the V_(H) -binding andF_(C) -binding segments) of a subject chimeric polypeptide can be eithercontiguous or adjacent to each other within the polypeptide chain. Wherethey are adjacent, the segments are separated by amino acid residuesforming a spacer segment typically comprised of from about 5conveniently up to about 50 residues, preferably about 15 to about 30residues as are found in Protein A and Protein G. A subject chimericpolypeptide can contain a plurality of the same or different V_(H)-binding and F_(C) -binding segments. Where three or more of theIg-binding segments are adjacent within a subject chimeric polypeptide,the spacer segments can be the same or different. It is preferred thatthe amino acid residue sequence of a spacer segment correspond to atleast a portion of the sequence of a spacer segment present in anaturally occurring F_(C) -binding protein, such as Protein A or ProteinG. A spacer segment can also be comprised of a sequence of residuecorresponding to a portion of the CD4 sequence that is contiguous to oneof the V_(H) -binding regions of CD4 as described herein.

A subject chimeric polypeptide can further contain a head and/or tailsegment of 1 conveniently up to about 50, such as about 5 orout 10,typically about 15 or about 30,at its amino- or carboxy terminus,respectively, where such a segment is advantageous in the polypeptide'smaking or use. For instance, a tail segment can provide a means forlinking the subject chimeric polypeptide to a solid matrix, where as aleader segment can advantageously be used to facilitate secretion of thepolypeptide during its expression in cells. It is preferred that theamino acid residue sequence of a head or tail segment found in Protein Aor Protein G. A head or tail segment can also be comprised of a sequenceof residues corresponding to a portion of the CD4 sequence that iscontiguous to one of the V_(H) -binding regions of CD4 as describedherein.

D. DNA and Recombinant DNA Molecules

In living organisms, the amino acid residue sequence of a protein orpolypeptide is directly related via the genetic code to thedeoxyribonucleic acid (DNA) sequence of the gene that codes for theprotein. Thus, a gene can be defined in terms of the amino acid residuesequence, i.e., protein or polypeptide, for which it codes.

An important and well known feature of the genetic code is itsredundancy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. Occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

The present invention contemplates a deoxyribonucleic acid (DNA)molecule or segment that defines a gene coding for, i.e., capable ofexpressing, a subject chimeric polypeptide. Preferred DNA molecules codefor chimeric polypeptides having a V_(H) -binding segment whose aminoacid residue sequence corresponds to a sequence shown in Tables 1 and 4.Preferred DNA molecules also include those coding for a F_(C) -bindingsegment whose amino acid residue sequence corresponds, and is preferablyidentical, to a sequence shown in Table 2. DNA molecules containingV_(H) -binding or F_(C) -binding segment-coding nucleotide sequencescorresponding to all or a portion of those shown in FIGS. 1-3 are mostpreferred.

DNA molecules that encode the subject proteins can easily be synthesizedby chemical techniques, for example, the phosophotriester method ofMatteucci et al., J. Am. Chem. Soc., 103:3185 (1981). Of course, bychemically synthesizing the coding sequence, any desired modificationscan be made simply by substituting the appropriate bases for thoseencoding the native amino acid residue sequence. However, DNA moleculesincluding base sequences identical to all or a portion of those shown inFIGS. 1-3 are preferred.

A DNA molecule that includes a DNA sequence encoding a subjectpolypeptide can be prepared by operatively linking (ligating)appropriate restriction fragments from each of the above depositedplasmids using well known methods. The DNA molecules of the presentinvention produced in this manner typically have cohesive termini, i.e.,"overhanging" single-stranded portions that extend beyond thedouble-stranded portion of the molecule. The presence of cohesivetermini on the DNA molecules of the present invention is preferred.

Also contemplated by the present invention are ribonucleic acid (RNA)equivalents of the above described DNA molecules.

The present invention further contemplates a recombinant DNA moleculecomprising a vector operatively linked, for replication and/orexpression, to a subject DNA molecule, i.e., a DNA molecule defining agene coding for a subject chimeric polypeptide.

As used herein, the term "vector" refers to a DNA molecule capable ofautonomous replication in a cell and to which another DNA segment can beoperatively linked so as to bring about replication of the attachedsegment. Vectors capable of directing the expression of a gene deliveredby a subject DNA segment are referred to herein as "expression vectors".Thus, a recombinant DNA molecule (RDNA) is a hybrid DNA moleculecomprising at least two nucleotide sequences not normally found togetherin nature.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed, these being limitations inherent in the art ofconstructing recombinant DNA molecules. However, a vector contemplatedby the present invention is at least capable of directing thereplication, and preferably also expression, of the subject chimericpolypeptide gene included in DNA segments to which it is operativelylinked.

In preferred embodiments, a vector contemplated by the present inventionincludes a procaryotic relicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a procaryotic host cell, such as abacterial host cell, transformed therewith. Such replicons are wellknown in the art. In addition, those embodiments that include aprocaryotic replicon also include a gene whose expression confers drugresistance to a bacterial host transformed therewith. Typical bacterialdrug resistance genes are those that confer resistance to ampicillin ortetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of the subject chimeric polypeptide gene in a bacterialhost cell, such as E. coli, transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment of the present invention. Typical of such vector plasmids arepUCs, pUCs, pBR322 and pBR329 available from Biorad Laboratories,(Richmond, Calif.) and pPL and pKK223 available from Pharmacia,Piscataway, N.J.

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1pML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC,#31255).

In preferred embodiments, the eucaryotic cell expression vectors used toconstruct the recombinant DNA molecules of the present invention includea selection marker that is effective in an eucaryotic cell, preferably adrug resistance selection marker. A preferred drug resistance marker isthe gene whose expression results in neomycin resistance, i.e., theneomycin phosphotransferase (neo) gene. Southern et al., J. Mol. Appl.Genet., 1:327-341 (1982).

The use of retroviral expression vectors to form the rDNAa of thepresent invention is also contemplated. As used herein, the term"retroviral expression vector" refers to a DNA molecule that includes apromoter sequence derived from the long terminal repeat (LTR) region ofa retrovirus genome.

In preferred embodiments, the expression vector is typically aretroviral expression vector that is preferably replication-incompetentin eucaryotic cells. The construction and use of retroviral vectors hasbeen described by Sorge, et al., Mol. Cell. Biol., 4:1730-37 (1984).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted and tothe vector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymer tracts can be added to theDNA segment to be inserted and to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase of E. coli DNApolymerase I, enzymes that remove protruding, 3', single-strandedtermini with their 3'-5' exonucleotytic activities and fill in recessed3' ends with their polymerizing activities. The combination of theseactivities therefore generates blunt-ended DNA segments. The blunt-endedsegments are then incubated with a large molar excess of linkermolecules in the presence of an enzyme that is able to catalyze theligation of blunt-ended DNA molecules, such as bacteriophase T4 DNAligase. Thus, the products of the reaction are DNA segments carryingpolymeric linker sequences at their ends. These DNA segments are thencleaved with the appropriate restriction enzyme and ligated to anexpression vector that has been cleaved with an enzyme that producestermini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies, Inc., New Haven, Conn.

Also contemplated by the present invention are RNA equivalents of theabove described recombinant DNA molecules.

The present invention also relates to a host cell transformed with arecombinant DNA molecule of the present invention preferably an RDNAcapable of expressing a subject chimeric polypeptide. The host cell canbe either procaryotic or eucaryotic. Bacterial cells are preferredprocaryotic host cells and typically are a strain of E. coli such as,for example, the E. coli strain DH5 available from Bethesda ResearchLaboratories, Inc., Bethesda, Md. Preferred eucaryotic host cellsinclude yeast and mammalian cells, preferably vertebrate cells such asthose from a mouse, rat, monkey or human fibroblastic cell line.Preferred eucaryotic host cells include Chinese hamster ovary (CHO)cells available from the ATCC as CCL61 and NIH Swiss mouse embryo cellsNIH/3T3 available from the ATCC as CRL 1658. Transformation ofappropriate cell hosts with a recombinant DNA molecule of the presentinvention is accomplished by well known methods that typically depend onthe type of vector used. With regard to transformation of procaryotichost cells, see, for example, Cohen et al., Proc. Natl. Acad. Sci. USA,69:2110 (1972); and Maniatis et al., Molecular Clonin A LaboratoryMammal, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).With regard to transformation of vertebrate cells with retroviralvectors containing rDNAs, see, for example, Sorge et al., Mol. Cell.Biol., 4:1730-37 (1984); Graham et al., Virol., 52:456 (1973); andWigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979).

Successfully transformed cells, i.e., cells that contain a recombinantDNA molecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anRDNA of the present invention can be cloned to produce monoclonalcolonies. Cells from those colonies can be harvested, lysed and theirDNA content examined for the presence of the RDNA using a method such asthat described by Southern, J. Mol. Biol., 98-503 (1975) or Berent etal., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of RDNA, successfultransformation can be confirmed by well known immunological methods whenthe RDNA is capable of directing the expression of a subject chimericpolypeptide. For example, cells successfully transformed with anexpression vector produce proteins displaying CD4 V_(H) -binding regionantigenicity. Samples of cells suspected of being transformed areharvested and assayed for the presence of CD4 V_(H) -binding regionantigenicity using antipolypeptide antibodies specific for that region.

Thus, in addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains a protein displaying antigen independent V_(H) -bindingactivity.

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources. Inembodiments wherein the host cell is mammalian, a "serum-free" medium ispreferably used.

E. Compositions and Polypeptides Linked to Labels and Solid Matricies

The present invention further contemplates a composition containing, inadmixture, a V_(H) -binding polypeptide, preferably a V_(H) -bindingpolypeptide having an amino acid residue sequence shown in Tables 1 and3, and a F_(c) -binding polypeptide, preferably a F_(c) -bindingpolypeptide having an amino acid residue sequence corresponding to thatof Protein A, Protein G, Protein Z and/or Table 2. Preferredcombinations include one or more of p16-49, p21-49, p16-38 and p21-38with one or more of Protein A, Protein G and Protein Z.

Typically, subject composition has a V_(H) -binding polypeptide to F_(c)-binding polypeptide molar ration in the range of about 1:10 to about10:1, preferable about 1:5 to about 5:1, and more preferably about 1:1.The admixed polypeptides can be in the form of a powder or solution.Solutions of V_(H) -binding and F_(c) -binding polypeptides typicallyhave a pH value range of about 5 to about 9 and an ionic strength of nomore than that equivalent to about one molar sodium chloride.

The V_(H) -binding polypeptides of the present invention, including thebefore described chimeras, can be linked to a label to form a labeledprobe. Preferred labels include alkaline phosphatase [O'Sullivan et al.,FEBS Letters, 95:311 (1978)], biotin, horse radish peroxidase,dichlorotriazinylaminofluorescein [DTAF; Blakeslee et al., J. Immunol.Meth., 13:320 (1977)], ferritin (Carlsson et al, Biochem. J., 173:723(1978)], fluoroscene isothiocyanste [FITC; McKinney et al., Anal.Biochem, 14:421 (1966)], beta-galactosidase [Ishikawa et al., Scand. J.Immunol., 8:43 (1978)], sulforhodamine 101 acid chloride (Texas Red),tetramethyrhodamine isothiocyanate [TRITC; Amante et al., J. Immunol.Meth., 1:289 (1972)], gold [Horisberger et al., Histochem., 82:219(1985)], and the like.

A V_(H) -binding polypeptide of this invention can be linked to a solidmatrix, i.e., a matrix substantially insoluble in aqueous solution,thereby forming an affinity sorbent useful in practicing the methods ofthis invention. Typical solid matrices include latex or acrylic beads,agarose, cellulose, SEPHAROSE (agarose beads) and the like. Preferably,the solid matrix is hydrophilic.

In view of the finding that complexes containing antibodies bound to apolypeptide of this invention display an increased affinity for antigen,such complexes in substantially isolated (substantially free ofantigen-antibody immunoreaction products) form are contemplated herein.Preferred V_(H) -binding polypeptide-antibody molecule complexes arethose containing a V_(H) -binding polypeptide having an amino acidresidue sequence corresponding, and preferably identical to, apolypeptide shown in Table 4. Preferably, only one type of antibodymolecule is present in the composition, i.e., a composition having thecharacteristics of a monoclonal antibody. The V_(H) -binding polypeptideis linked to the V_(H) region of the antibody molecule at a site otherthan the antibody molecule's antigen-binding site. The complex istherefore capable of immunoreacting with antigen, or, in the case ofcatalytic antibody molecules (abzymes), binding substrate as part of anenzymatic reaction. The composition can take the form and be formulatedas previously discussed for the subject polypeptides.

In another embodiment, the present invention contemplates a V.-bindingconjugate comprised of a V_(H) -binding polypeptide of this inventionoperatively linked, preferably covalently, to an F_(C) -bindingpolypeptide, the linkage being by other than a peptide bond between thealpha-amino group and carboxy group of contiguous amino acid residues.In preferred conjugates, one or more of the peptides of Table 4 arecoupled to Protein A, Protein G, Protein Z or an F_(C) -binding peptideof Table 2.

Also contemplated is a _(V) H-binding self-conjugate comprised of aplurality of V_(H) -binding polypeptides operatively linked, preferablycovalently, to each other by other than a peptide bond between thealpha-amino group and carboxy group of contiguous amino acid residues.Preferred self-conjugates contain a V_(H) -binding peptide of Table 4.one type of self-conjugate is a homoconjugate wherein each of theplurality of linked peptides is substantially identical in amino acidresidue sequence.

In preferred embodiments, the V_(H) -binding conjugates of thisinvention are in a composition substantially free of, or isolated from,non-conjugated (free) V_(H) -binding polypeptide so that of the totalpeptide present, less than about 5% by weight is in non-conjugated formof course, the conjugates and conjugate compositions of this inventioncan take the form and be formulated as previously discussed for thesubject polypeptides.

F. Methods

The affinity of the subject V_(H) -binding polypeptides for Ig canadvantageously be used in any method similar to that reported forProtein A, Protein G or Protein Z. Particularly preferred is a methodfor separating Ig molecules, including V_(H) -containing portions of Igmolecules, from an aqueous composition containing a complex mixture ofproteins and recovering the separated Ig molecule and/or Ig-depletedcomposition as a purified product. See, for example, U.S. Pat. Nos.4,409,330, No. 4,464,165 and No. 4,687,734. Generally, the methodutilizes the following steps.

(1) Admixing the Ig-containing (V_(H) -containing) aqueous sample with asubject polypeptide to form a binding reaction admixture. The Igmolecules can be present in the sample in free (nonimmunologicallybound) form or in the form of immune complexes.

(2) Maintaining the binding reaction admixture under Ig-bindingconditions for a time period, typically predetermined, sufficient for aportion of the Ig molecules present to bind the subject polypeptide,thereby forming a Ig-polypeptide complex and a non-bound sample portion.Maintenance time periods are typically in the range of about 10 minutesto about 16-20 hours under Ig-binding conditions.

"Ig-binding conditions" are those that maintain the biological activityof the polypeptide molecules of this invention and the antibodies soughtto be bound, and include a temperature range of about 4 degrees C. toabout 45 degrees C., a pH value range of about 5 to about 9 and an ionicstrength of no more than that equivalent to about one molar of sodiumchloride. Methods for optimizing such conditions are well known in theart.

(3) Segregating the complex from the non-bound sample portion andrecovering the segregated complex as isolated Ig and/or recovering thesegregated non-bound sample portion as Ig-depleted sample. Segregationof reactants from products is typically accomplished using a sizeseparation technique, such as centrifugation, chromatography and thelike, and/or washing, such as by dialyzing, with an aqueous solution,preferably a buffer, that does not significantly promote dissociation ofthe complex, of course, the Ig can also be separated and recovered fromthe segregated complex by adaptation of any of the techniques similarlyused in the art for Protein A, Protein G and the like. Such methodstypically involve exposing the complex to a destabilizing saltconcentration (usually low relative to the Ig-binding condition used)and/or pH value (also usually low relative to the Ig-binding conditionused). Competitive dissociation with an agent such as dextran sulfatecan also be employed, typically followed by dialization to remove thecompetitive agent.

The affinity of the subject V.-binding polypeptides can also be used toassay for the presence of V_(H) molecules, typically in the form ofwhole Ig and/or V_(H) -containing portions thereof, in a sample. Themethod involves admixing a sample suspected of containing V_(H)molecules with a subject V_(H) -binding polypeptide. The bindingreaction admixture so formed is maintained under Ig-binding conditionsas previously described to permit complex formation between the subjectV_(H) -binding polypeptide and any V. molecules present in the sample.The presence of any complex formed is then detected, and thereby thepresence of V_(H) molecules present in the sample.

Methods for detecting the presence and/or amount of the V_(H) -bindingpolypeptide-containing complex formed are well known and include thoseused in the art for detecting complexes containing Protein A and ProteinG. A preferred method involves detecting the presence in the complex ofa label that has been operatively linked to the subject V_(H) -bindingpolypeptide, either before or after complex formation.

The present invention further contemplates a method of increasing theaffinity of an antibody for an antigen with which it immunoreacts. Themethod comprises forming a complex containing the antibody and a V_(H)-binding polypeptide of this invention, preferably a polypeptideconsisting essentially of a V_(H) -binding fragment of CD4, and morepreferably one or more of peptides shown in Table 4. The V_(H) -bindingpolypeptide can be covalently linked to the V_(H), but the Methodtypically first involves forming a non-covalent complex via thepreviously discussed binding reaction.

EXAMPLES

The following examples illustrate but do not limit the presentinvention.

1. Peptide D16-49 of CD4 Binds to Murine Monoclonal Antibodies and HumanMyeloma Proteins

A. Polypeptides

The polypeptides, whose amino acid residue sequences are shown in Tables1 and 4, were synthesized using the classical solid-phase techniquedescribed by Merrifield, Adv. Enzymol., 32:221-96 (1969) as adapted foruse with a model 430A automated peptide synthesizer (Applied Biosystems,Foster City, Calif.). Polypeptide resins were cleaved by hydrogenfluoride, extracted and analyzed for purity by high-performance liquidchromatography (HPLC) using a reverse-phase C18 column manufactured byWaters Associates, Milford, Mass.

B. Peptide P21-49 Binds Murine and Human Antibodies.

The binding of peptide p21-49 to the mouse and human antibodies wasexamined by Enzyme-Linked Immuno-absorbent Assay (ELISA) in a mannersimilar to that described in Antibodies A Laboratory Manual, Harloe andLane, Cold Spring Harbor Laboratory, N.Y., 1988. Briefly, solid-phaseaffixed p21-49 (a p21-49-containing solid support) was prepared byadmixing 50 microliters (ul) of 0.9% sodium chloride (coating buffer)containing 5 to 10 micrograms (ug) per milliliter (ml) of the peptide tothe wells of a polyvinyl or polystyrene microtiter plate. The plate wasmaintained for six hours at 37 degrees (37° C.) on a rotating platformto allow the peptide to adhere to the wells and form solid supports.After aspirating the excess liquid from the wells, 200 ul of washingsolution containing TWEEN 20 (0.5% polyoxyethylenesorbitan monolaurate)in phosphate buffered saline (0.5M sodium chloride, 0.01M sodiumphosphate at pH 7.2; PBS) was admixed to each well and immediatelyremoved by aspiration. After aspirating the excess liquid from the wells200 ul of blocking solution consisting of 1% bovine serum albumin (BSA)in phosphate buffered saline at pH 7.4 was admixed to each well, and thewells maintained at room temperature for one hour. The wells were washedwith 200 ul a solution containing 0.5% TWEEN-20 (Polyoxyethylenesorbitanmonolaurate) in PBS and the wash solution immediately removed byaspiration.

A first antibody, either a murine monoclonal antibody or a purifiedhuman myeloma protein was diluted to approximately 80 ug per ml inbinding buffer consisting of 1% BSA and 0.5% TWEEN 20(polyoxyethylenesorbitan monolaurate) in PBS and then various amounts,depending on the final antibody concentration desired, were admixed toeach well and the wells maintained at room temperature for 3 or 4 hoursin a humidified atmosphere. The wells were then washed 6 times withwashing buffer and the excess liquid removed by aspiration.

50 ul of a horse radish peroxidase (HRP)-conjugated anti-mouse Igsecondary antibody (Cappel, Cochranvile, Pa.) or HRP-conjugatedantihuman Ig (Sigma, St. Louis, Mo.) diluted 1:10,000 and 1:1,500,respectively, in binding buffer supplemented with 15% heat inactivatedcalf serum, was then admixed to each well. The wells were maintained for1 hour at room temperature and then washed several times with washingbuffer. 100 ul of freshly prepared substrate, prepared by adding 4 mg of0-phenylendiamine and 4 ul of a 30% solution of hydrogen peroxide to 10ml of a buffer containing 0.1M citric acid and 0.2M dibasic sodiumphosphate at pH 5.0. were admixed to each well. The wells were thenmaintained (incubated) for 30 minutes at room temperature in the dark.The reaction was stopped by adding 25 ul per well of a stop bufferconsisting of aqueous 4N H₂ SO₄. The amount of colored reaction productproduced was determined by measuring the absorbance at 492 nanometers(nm).

The binding of peptide p21-49 to 22 murine monoclonal antibodies ofdifferent antigen specificities and isotypes (groups 1, II and III) isshown in FIG. 4A. Peptide p21-49 binds murine monoclonal antibodiesimmunospecific for 3 different antigens demonstrating that the peptidebinds antibody regardless of that antibody's specificity.

The binding of peptide p21-49 to 36 human myeloma proteins of differentisotypes is shown in FIG. 4B. Peptide p21-49 binds human myelomaproteins of 8 different isotypes demonstrating the peptide'sisotype-independent antibody binding properties.

The isotype and specificity of each of the antibodies tested are listedin Table 3.

                  TABLE 3                                                         ______________________________________                                        Antibody                                                                      Group       Antibody    Antibody                                              (Panel/#)   Isotype     Specificity                                           ______________________________________                                        A/I         IgG.sub.1,IgM                                                                             bovine thyroglobulin                                  A/II Factor IgG r       human Von Willebraud                                  A/III       IgG,IgG.sub.2b                                                                            nitrophenyl acetyl                                    B/I         IgG.sub.1   human myeloma                                         B/II        IgG.sub.2   human myeloma                                         B/III       IgG.sub.3   human myeloma                                         B/IV        IgG.sub.4   human myeloma                                         B/V         IgM         human myeloma                                         B/VI        IgA         human myeloma                                         B/VII       IgD         human myeloma                                         B/VIII      IgE         human myeloma                                         ______________________________________                                    

2. Peptide P21-49 Binds Specifically to Immunoglobulins.

The specificity of the binding interaction between peptide P21-49 andimmunoglobulins was determined by inhibiting that interaction withliquid phase peptide p21-49. In addition to demonstrating the bindingspecificity of this peptide, the apparent binding constant was alsodetermined. After the microtiter wells were coated with peptide P21-49as described in Example 1B using peptide at a concentration of 20 ug/mlin coating buffer, peptide p21-49 was added to three replicate wells ata final concentration of 40 ug/ml, 20 ug/ml, 10 ug/ml, 5 ug/ml, 2.5ug/ml, 1.25 ug/ml, 0.62 ug/ml and 0.31 ug/ml. An aliquot (25 ul) ofHRP-conjugated murine monoclonal antibody, diluted to about 1 ug/ml inbinding buffer, was then admixed to each well. The wells were maintainedat room temperature for 3 or 4 hours in a humidified atmosphere. Thewells were then washed 6 times with washing buffer and the excess liquidremoved by aspiration. The amount of monoclonal antibody bound in eachwell was then determined as described in Example 1B.

Monoclonal antibody 62, monoclonal antibody 18.85, monoclonal antibodyQ5/13 and monoclonal antibody 18.1.16 were studied in this fashion andthe percent of maximal binding versus the peptide concentration wasfound to be about linear over the peptide concentration range examinedfor each MAb, as is shown in FIG. 5. The apparent binding constants inml per gram are also shown for each of the monoclonal antibodies in FIG.5. It should be noted that the binding constants, which were all in therange of 3-5×10⁵ ml/g, are somewhat less than is typically observed formonoclonal antibody-antigen immunoreactions, which are typically on theorder of 10⁶ -10¹² ml/g.

3. Localization of the CD4 Region Containing the Immunoglobulin BindingActivity.

The region of CD4 that binds immunoglobulins was localized bysynthesizing, according to Example 1A, the peptides shown in Table 4.Each peptide's ability to bind mouse monoclonal antibodies wasdetermined using the ELISA assay described in Example 1B.

                                      TABLE 4                                     __________________________________________________________________________    CD4 PEPTIDES                                                                  Ig                                                                            Binding.sup.1                                                                      Designation.sup.2                                                                    SEQUENCE                                                          __________________________________________________________________________    4+   p 16-49                                                                              CTASQ KKSIQ FHWKN SNQIK ILGNQ GSFLT KGPS                          4+   p 21-49                                                                              KKSIQ FHWKN SNQIK ILGNQ GSFLT KGPS                                3+   p 16-38                                                                              CTASQ KKSIQ FHWKN SNQIK ILG                                       3+   p 21-38                                                                              KKSIQ FHWKN SNQIK ILG                                             *    p 21-37                                                                              KKSIQ FHWKN SNQIK IL                                              *    p 21-36                                                                              KKSIQ FHWKN SNQIK I                                               *    p 21-35                                                                              KKSIQ FHWKN SNQIK                                                 0    p 21-34                                                                              KKSIQ FHWKN SNQI                                                  *    p 21-33                                                                              KKSIQ FHWKN SNQ                                                   *    p 22-38                                                                              KSIQ FHWKN SNQIK ILG                                              *    p 23-38                                                                              SIQ FHWKN SNQIK ILG                                               *    p 24-38                                                                              IQ FHWKN SNQIK ILG                                                0    p 21-29                                                                              KKSIQ FHWK                                                        2+   p 25-38                                                                              Q FHWKN SNQIK ILG                                                 2+   p 29-43                                                                              KN SNQIK ILGNQ GSF                                                1+   p 32-54                                                                              NQIK ILGNQ GSFLT KGPSK LNDR                                       1+   p 38-62                                                                              GNQ GSFLT KGPSK LNDRA DSRRS LW                                    0    p 39-60                                                                              NQ GSFLT KGPSK LNDRA DSRRS                                        0    p 42-49                                                                              SFLT KGPS                                                         0    p 48-72                                                                              PSK LNDRA DSRRS LWDQG NFPLI IK                                    1+   p 58-82                                                                              RRS LWDQG NFPLI IKNLK IEDSD TY                                    1+   p 66-90                                                                              NFPLI IKNLK IEDSD TYICE YEDQK                                     0+   p 137-161                                                                            NIQG GKTLS VSQLE LQDSG TWTCT Y                                    1+   p 128-161                                                                            VQC RSPRG KNIQG GKTLS VSQLE LQDSG TWTCT Y                         __________________________________________________________________________     .sup.1 The amount of antigenindependent immunoglobulin binding activity       detected for each peptide is shown on a scale of 0 to 4+ with 0 being no      substantial binding, 1+ being weak binding and 2+, 3+ and 4+ indicating       increasing levels of significant binding.                                     .sup.2 The designation given each peptide corresponds to the position of      the peptide's sequence within the CD4 molecule as shown in FIG. 1.            *See FIG. 15 for relative binding affinity of this peptide.              

The results shown in Table 4 and FIG. 15 indicate that the amino acidresidue sequence IKIL, corresponding to residues 34-37 of CD4, appearsto play an important role in the antigen-independent binding ofpolypeptide to V_(H). Peptides p22-38, p23-38, p24-38 and p21-37 gavecomparable binding with that of p21-38 and p21-49. Peptides 21-36 and21-35 bound with the above background values.

4. Peptide P21-49 Exhibits Enhanced Binding to Antigen-AntibodyComplexes

The effect of antigen binding on peptide p21-49 immunoglobulin bindingwas determined using monoclonal antibodies immunospecific forthyroglobulin. Briefly, p21-49 was affixed to the wells of microliterplates as described in Example 1B. Thyroglobulin was then admixed to thewells at a final concentration ranging from 0.005 mg/ml to 4 mg/ml.Monoclonal antibodies immunospecific for thyroglobulin were then admixedto the wells at a concentration of approximately 20 ug/ml in bindingbuffer consisting of 1% BSA and 0.5% Tween-20 in PBS and the wellsmaintained at room temperature for 4 hours in a humidified atmosphere toallow the antigen-antibody complexes to form and to be bound by theimmobilized peptide. The wells were then washed 6 times with washingbuffer and the excess liquid removed by aspiration. The assay wascompleted according to Example 1B.

At final concentrations of greater than 0.5 ug/ml, thyroglobulinenhanced the binding of the antibody to the p21-49 immobilized on theplate as shown in FIG. 6A. These results suggest that the binding ofantigen by an antibody induces a conformational change in the antibodywhich in turn increases the affinity of the antibody for the V_(H)-binding polypeptide.

In a similar assay designed to show that V_(H) -binding polypeptides ofthis invention do not interfere with an antibody's ability to bindantigen, thyroglobulin was affixed to microtiter wells as described inExample 1B using 50 ul of coating buffer containing 2 ug/mlthyroglobulin. Peptide pl6-49 was then admixed to each well at a finalconcentration ranging from 0.015 ug/ml to 40 ug/ml. Theanti-thyroglobulin murine monoclonal antibody, diluted to approximately20 ug/ml in binding buffer consisting of 1% BSA and 0.5% Tween-20 inPBS, was admixed to each well and the wells maintained at roomtemperature for 4 hours in a humidified atmosphere. 200 ul of washingsolution was admixed to each well and removed by aspiration and theremainder of the assay completed according to Example 1B.

Peptide pl6-49 did not interfere with antigen binding even at aconcentration of 40 ug/ml as shown in FIG. 6B. Thus, the polypeptides ofthe present invention do not bind Ig at the Ig antigen binding site.

5. Binding of Peptide v21-49 to Immunoglobulins is Inhibited by DextranSulfate.

The binding of peptide p21-49 to various immunoglobulins was determinedin the presence of varying concentration of dextran sulfate. Briefly,peptide p21-49 was affixed to the wells of microliter plates asdescribed in Example 1B. Dextran sulfate was then admixed to individualwells at a final concentration ranging from 0.01 ug/ml to 50 ug/ml. Thefirst antibody, either a murine monoclonal antibody immunospecific forthyroglobulin or a murine monoclonal antibody immunospecific for CD4protein was diluted to approximately 4 ug/ml in binding buffer andadmixed to each well. The wells were maintained at room temperature for4 hours in a humidified atmosphere. The wells were then washed 6 timeswith washing buffer and the excess liquid removed by aspiration. Theassay was completed according to Example 1B.

The inhibition of peptide p21-49 binding to immunoglobulin in thepresence of various concentrations of dextran sulfate is shown in FIG.7. Dextran sulfate inhibits the binding of peptide p21-49 to both amonoclonal antibody immunospecific for thyroglobulin (62) and amonoclonal antibody immunospecific for CD4 (OKT4A).

6. Characterization of the CD4-Binding Site on Immunoglobulins.

A series of ELISA assays were performed to localize the peptide p21-49binding site on immunoglobulin molecules. These assays were similar tothe assay described in Example 1B, with the exceptions noted below.Here, the assay described in Example 1B was carried out using isolatedimmunoglobulin heavy chains instead of the first monoclonal antibody.The isolated immunoglobulin heavy chains were prepared according to themethods described in Zanetti et al, J. Immunol., 135:1245 (1985). Thepeptide p21-49 bound isolated heavy chain about as well as intactantibody as shown in FIG. 8A.

A similar assay was carried out using isolated immunoglobulin lightchain prepared according to the methods described in Zanetti et al.,supra, instead of the first monoclonal antibody. The results of thisassay, also shown in FIG. 8A, indicate that the light chain did notcontribute to immunoglobulin binding by the peptide p21-49.

Further evidence that the binding site for peptide p21-49 was localizedto the heavy chain was provided by assays carried out using recombinantchimeric (mouse/human) antibodies of the IgG₁ (human) isotype withdifferent isotypes of light chain (kappa and lambda). The assay wascarried out according to Example 1B except that the first monoclonalantibody was replaced by either recombinant chimeric IgG₁ -lambda orrecombinant chimeric IgG₁ -kappa antibodies. The assay was completedaccording to Example 1B and as shown in FIG. 8B, changing the lightchain isotype has no effect on the binding of peptide p21-49 to the IgG₁heavy chain. The assays with isolated heavy chain, isolated light chain,and the recombinant chimeric antibodies localize the peptide p21-49binding to the heavy chain.

To determine whether peptide p21-49 bound the immunoglobulin heavy chainvariable region (V_(H)) or the immunoglobulin heavy chain Fc region, aseries of assays utilizing immunoglobulin molecules containing only thevariable region [F(ab')₂ Fab] were carried out according to Example 1Bexcept that molecules containing only variable regions were used insteadof the first monoclonal antibody. Both types of molecules containingonly the variable regions [(Fab')₂ and Fab] bound peptide p21-49indicating that the peptide binds somewhere in the variable region ofthe heavy chain (FIG. 8C).

Further assays were performed using naturally occurring deletion mutantsof MOPC 21, a murine myeloma protein of unknown specificity, that havedeleted either the CH₃ domain or the CH₁ domain of the Ig V_(H). Theassays were carried out according to Example 1B except that the mutantantibodies were used instead of the monoclonal antibody. Neitherdeletion in the CH₃ domain (residues 358-440, Sequences of Proteins ofImmunological Interest, Kabat et al., U.S. Dept. of Health and HumanServices, 4 ed, 1987) nor a deletion in the CH₁ domain (residues121-214, Sequences of Proteins of Immunological Interest, Kabat et al.,U.S. Dept. of Health and Human Services, 4 ed, 1987) diminished thebinding of peptide p21-49 to the immunoglobulin molecules as shown inFIG. 8D. Therefore, peptide p21-49 binds the V_(H) region ofimmunoglobulins.

8. Purification of Immunoglobulins Using Peptide P21-49.

The immunoglobulins were purified from an immunoglobulin containingsample using a peptide p21-49 affinity column. The affinity column wasprepared by coupling 5 mg of peptide p21-49 to 1 ml of hydratedSepharose 4B-CNBr activated resin. The peptide was coupled to theSepharose 4B-CNBr resin according to the manufacturers directions(Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.). Briefly one gramof sepharose 4B-CNBr activated resin was dissolved in 1 mm HCl andmaintained for two hours at room temperature. The Sepharose 4B-CNBr wasrinsed with the same buffer on a sintered glass filter and thenneutralized with 0.1M carbonate buffer at pH 8.3 containing 0.5M NaCl.After the activated Sepharose 4B was neutralized, the resin wascollected by centrifugation and peptide p21-49 at a final concentrationof 10 mg/ml in 0.1M carbonate buffer at pH 8.3 containing 0.5M NaCl wasadmixed to the activated resin for 14 hours at 4C. The resin was washedwith PBS and the remaining active groups on the Sepharose 4B blockedwith glycine by adding glycine to a final concentration of 0.2M at pH8.0 and maintaining the solution for 2 hours at room temperature. Theexcess blocking glycine was then washed away with PBS and the coupledresin treated with 0.1M glycine buffer at pH 2.5 followed by treatmentwith PBS at pH 7.5 containing 0.1% NaN₃. The peptide-Sepharose 4Bconjugate was stored at 4C until used.

A semi-purified immunoglobulin sample (saturated ammonium sulfatefraction of ascites fluid containing 1 to 2 mg of mouse immunoglobulin)in PBS at PH 7.4 was loaded onto a column containing approximately 1 mlof the packed peptide-Sepharose 4B conjugate resin. The semi-purifiedsample was allowed to flow slowly through the column allowing theimmunoglobulin present in the sample to be bound (segregated into asolid phase) by the immobilized peptide p21-49. After the sample wasapplied to the column, the column was washed with 2 ml of PBS at pH 7.4to further segregate the bound immunoglobulins from other non-boundproteins. The bound immunoglobulin was eluted with either 2 ml of 100 mmcitric acid at pH 3.0 or a PBS buffer containing dextran sulfate at afinal concentration of 50 ug/ml at pH 7.4.

The initial recovery of separated (isolated) immunoglobulin, illustratedin FIG. 14, was about 65% of the applied semi-purified immunoglobulinsample. The isolated immunoglobulin retained all its major biologicalfunctions including antigen binding, complement fixation, and its puritywas calculated to be greater than 95% pure using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS PAGE).

9. Co-expression of V. Binding Peptide p21-49 with StaphylococcalProtein A F,-Binding Region.

A V_(H) -binding protein is expressed with a staphylococcal Protein AF_(C) -binding region by operatively linking a synthetic DNA sequence toa vector designed to express foreign proteins as a fusion protein withthe staphylococcal protein A F_(C) -binding region. The pRIT5 protein Agene fusion vector FIG. 9 is commercially available from Pharmacia LKB(Piscataway, N.J.) and is based on the methods and techniques in Nilssonet al., EMBO J., 1075-1080 (1985); and Uhlen et al., Gene, 23:369-378(1983). The pRIT5 protein A gene fusion vector is designed to permithigh-level expression of fusion proteins in both E. coli andStaphylococcus aureus host cells. A gene inserted into the multiplecloning site is expressed from the protein A promoter and translocatedto the periplasmic space in E. coli or secreted into the growth mediumin gram-positive cells such as Staphylococcus aureus. The protein Apromoter and signal sequence are functional in a wide variety ofbacterial species including S. aureus strain SA113 described in Uhlen etal., J. Bact., 159:713 (1984). The pRIT5 vector contains thebroad-host-range origin of replication and the chloramphenicol acetyltransferase gene from PC194 described in Horincuehi et al., J. Bact.,150:815 (1982).

A synthetic DNA sequence coding for the V_(H) -binding peptide p21-49with Bam HI restriction endonuclease (restriction enzyme) sites attachedto the ends is constructed by synthesizing the following polynucleotidesusing an Applied Biosystem DNA Synthesizer and following themanufactures instructions (Applied Biosystems, Foster City, Calif.).

01--GATCCCAAAAAAAAAGTATCCAATTCCATTGGAAAAACAGTAACAGTAACCAAAT

02--CAAAATCTTAGGTAACCAAGGTAGTTTCTTAACTAAAGGTCCTAGTG

03--AATGGAATTGGATACTTTTTTTTTG

04--TAAGATTTTGATTTGGTTACTGTTTTTCC

05--TGCAGACTAGGACCTTTCGTTAAGAAACTACCTTGGTTACC

These polynucleotides are designed to hybridize to each other and formthe double stranded sequence shown in FIG. 10.

Polynucleotides 02, 03, 03 and 04 are kinased by adding 1 ul of 100ug/ml solution of each of the polynucleotides, 20 units of T4polynucleotide kinase to a solution containing 70 mmtris(Hydroxymethyl]aminomethane (Tris-HCl) at pH 7.6, 10 mM MgCl₂ 5 mMdithiothreitol (DTT) , 10 MM 2-mercaptoethanol (2 ME), 500 mg/ml ofbovine serum albumin (BSA). The solution is maintained at 37° C. forabout 30 minutes and the reaction stopped by maintaining the solution at65C for 10 minutes. The two end polynucleotides, 20 ng of polynucleotide01 and 20 ug polynucleotide 05, are added to the above kinasing reactionsolution together with 1/10 volume of a solution containing 20.0 mMTris-HCl at pH 7.4, 2.0 MM MgCl₂ and 15.0 MM NaCl. This solution isheated to 70° C. for 5 minutes and allowed to cool to room temperature,approximately 25° C., over 1.5 hours in a 500 ml beaker of water. Duringthis time period all the polynucleotides anneal to form the doublestranded synthetic DNA insert. The individual polynucleotides arecovalently linked to each other to stabilize the synthetic DNA insert byadding 40 ul of the above reaction to a solution containing 50 mMTris-HCl at pH 7.5, 7 MM MgCl₂, 1 MM DTT, 1 MM ATP and 10 units of T4DNA ligase. This solution is maintained at 37° C. for 30 minutes andthen the T4 DNA ligase is inactivated by maintaining the solution at 65°C. for 10 minutes. The end polynucleotides are kinased by mixing 52 ulof the above reaction, 4 ul of a solution containing 10 MM ATP and 5units of T4 polynucleotide kinase. The solution is maintained at 37° C.for 30 minutes and then the T4 polynucleotide kinase is inactivated bymaintaining the solution at 65° C. for 10 minutes.

The completed synthetic DNA insert shown in FIG. 10, is ligated directlyinto the pRIT5 protein A gene fusion vector that has been previouslydigested with the restriction endonuclease Bam HI followed by alkalinephosphatase treatment to remove 5'-terminal phosphates. The ligationmixture is then transformed into suitable host cells and plated out toproduce single colony transformants. DNA is prepared from several of theresulting single colony transformants and the DNA sequence of thesynthetic DNA insert coding for the V_(H) -binding peptide determinedusing methods similar to or dependent on the chain termination DNAsequencing methodology described by Sanger et al., Proc. Natl. Acad.Sci., USA, 74:5463-5467 (1973). This DNA sequencing confirms theaccuracy of the above construction steps.

The protein A F_(C) -binding:V_(H) -binding peptide p21-49 fusionprotein is expressed in E. Coli and S. aureus using methods similar tothe methods described in Nilsson et al., EMBO J., 4:1075-1080 (1985);and Uhlen et al., Gene, 23:369-378 (1983).

The level of expression of the protein A F_(C) -binding:V_(H) -bindingpeptide p21-49 is determined using an ELISA assay capable of detectingthe immunoglobulin binding peptide p21-49 in host cell extracts and onhost cell membranes.

10. Co-expression of V.-binding Peptide P21-49 With StreptococcalProtein G F_(c) -binding Region.

The V_(H) -binding peptide p21-49 is expressed with streptococcalprotein G F_(C) -binding region by operatively linking a synthetic DNAsequence coding for the F_(C) -binding region of streptococcal G to oneof the vectors, shown in FIG. 11, containing the staphylococcal ProteinA promoter and signal sequence described in Abrahmsen et al., EMBO J.,4:3901-3906 (1985). The vector series, pAS1-3 is designed so that aforeign gene can be inserted directly following the staphylococcalprotein A signal sequence and thus the gene will be expressed as afusion protein with the staphylococcal protein A signal sequence at itsN-terminus. The pAS1-3 vector series is designed to permit high levelexpression of such fusion proteins in both E. coli and StaphylococcusAureus host cells. A gene inserted into the multiple cloning site isexpressed from the Protein A promoter and translated to the periplasmicin E. coli or secreted into the growth medium in gram-positive cellssuch as Staphylococcus aureus. The Protein A promoter and signalsequence are functional in a wide variety of bacterial species includingS. aureus strain SA113 described in Uhlen et al., J. Bact., 159:713(1984). The pAS 1-3 series of vectors contain both the origin ofreplication from E. coli and the origin of replication fromStaphylococci/Bacilli thus allowing the vectors to be grown in a widerange of host cells. The pAS 1-3 series of vectors also contain thechloramphenicol acetyl transferase gene from PC194 described inHorincuehi et al., J. Bact., 150:815 (1982).

A synthetic DNA sequence coding for the binding portion of streptococcalprotein G with a Eco RI restriction enzyme site on the ends isconstructed by synthesizing the following polynucleotides using anApplied Biosystems DNA Synthesizer and following the manufacturersinstructions (Applied Biosystems, Foster City, Calif.).

G1--AATTCATCGATGCGTCTGAATTAACACCAGCCGTGACAACT

G2--ACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACA

G3--CTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTT

G4--CAAACAATACGCTAACGACAACGGTGTTGACGGTGAATGG

G5--ACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAAA

G6--AACCAGAAGTGATCGATGGATCCG

G7--TTAATTCAGACGCATCGATG

G8--TTACCATTAATAACAAGTTTGTAAGTTGTCACGGCTGGTG

G9--AGCATCAACAGCTTCAGTAGTTGTTTCGCCTTTCAATGTT

G10--TGTCGTTAGCGTATTGTTTGAAGACTTTTTCTGCAGTAGC

G11--TAGTCGCATCGTCGTAAGTCCATTCACCGTCAACACCAT

G12--AATTCGGATCCATCGATCACTTCTGGTTTTTCAGTAACTGTAAAGGTCT

These polynucleotides are designed to hybridize to each other and formthe double stranded DNA sequence shown in FIG. 12.

Polynucleotides G2 through G11 are kinased by adding one ul of a 100ug/ml solution of each of the nucleotides, 20 units of T4 polynucleotidekinase to a solution containing 70 MM Tris-HCl at pH 7.6, 10 MM MgCl₂, 5mm DDT, 10 mm 2ME, 500 ug/ml of BSA. The solution is maintained at 37°C. for about 30 min and the reaction stopped by maintaining the solutionat 65° C. for 10 minutes. The two end polynucleotides 20 ng ofpolynucleotide G1 and polynucleotide G12 are added to the above kinasingreaction solution together with 1/10 volume of a solution containing 20MM Tris-HCl at pH 7.4, 2 MM MgCl₂ and 15 MM NaCl. This solution isheated to 70° C. for five minutes and allowed to cool to roomtemperature, approximately 25° C., over 1.5 hours in a 500 ml beaker ofwater. During this time all the polynucleotides anneal to form thedouble stranded synthetic DNA insert. The individual polynucleotides arecovalently linked to each other to stabilize the synthetic insert byadding 40 ul of the above reaction mixture to a solution containing 50MM Tris-HCl at pH 7.5, 7 MM MgCl₂, 1 mM DTT, 1 mm ATP and 10 units of T4DNA ligase. This solution is maintained at 37° C. for 30 minutes andthen the T4 DNA ligase is inactivated by maintaining the solution at 65°C. for 10 minutes. The end polynucleotides are kinased by mixing 52 ulof the above reaction, 4 ul of a solution containing 10 mm ATP and 5units of T4 polynucleotide kinase. The solution is maintained at 37° C.for thirty minutes and then the T4 polynucleotide kinase is inactivatedby maintaining the solution at 65° C. for 10 minutes.

The completed DNA insert is ligated directly into the pAS1 vector thathas been previously digested with the restriction enzyme Eco RI followedby treatment with alkaline phosphatase to remove 5'-terminal phosphategroups. The ligation mixture is then transformed into suitable hostcells and plated out to produce single colony transformants. DNA isprepared from several of the resulting single colony transformants andthe DNA sequence of the synthetic DNA insert coding for the Protein GF_(C) -binding region is determined using methods similar to, ordependent on the chain termination DNA sequencing methodology describedby Sanger et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467 (1973). ThisDNA sequencing confirms the accuracy of the above construction steps.

The synthetic DNA insert constructed above and shown in FIG. 12 containsa unique Bam HI restriction enzyme site. The new vector resulting fromthe above construction steps is linearized at this unique Bam HI siteand the synthetic DNA sequence coding for the V_(H) -binding peptidep21-49 constructed in Example 9 (FIG. 10) is ligated into this site.This ligation mixture is then transformed into suitable host cells andDNA is prepared from several of the resulting single colonytransformants. The DNA sequence of the two synthetic DNA inserts and theregions surrounding them are determined using the chain termination DNAsequencing methodology. The resulting vector is able to co-expressProtein G F_(C) -binding region and V_(H) -binding peptide p21-49 as afusion protein with the signal sequence from staphlacoccal Protein A.

The protein G F_(C) -binding region:V_(H) -binding peptide fusionprotein is expressed in E. coli and S. aureus using methods similar tothe methods described in Nilsson et al., EMBO J., 4:1075-1080 (1985);and Uhlen et al., Gene, 23:369-378 (1983).

The level of expression of the protein G F_(C) -binding:V_(H) -bindingpeptide is determined using an ELISA assay capable of detecting theimmunoglobulin binding peptide p21-49 in host cells extracts and on hostcell membranes.

11. Co-expression of V_(H) -binding peptide P21-49 With StaphylococcalProtein A and Streptococcal Protein G-F_(C) -binding Regions.

V_(H) -binding protein 21-49 is expressed with F_(C) -binding regionsderived from both Staphylococcal protein A and Streptococcal protein Gby operatively linking a synthetic DNA sequence containing the F_(C)-binding region of Streptococcal protein G to a vector designed toexpress foreign proteins as a fusion protein with the Staphylococcalprotein A F_(C) -binding region. The pRIT5 protein A gene fusion vector(FIG. 9) is commercially available from Pharmacia LKB (Piscataway, N.J.)and is based on the methods and techniques described in Nilsson et al.,EMBO J., 1075-1080 (1985); and Uhlen et al., Gene, 23:369-378 (1983). Asynthetic DNA sequence coding for the F_(C) -binding portion of proteinG with 5' overhanging ends compatible with a Bam HI restriction site wasconstructed by synthesizing the following polynucleotides using anApplied Biosystems DNA Synthesizer and following the manufacturersinstructions (Applied Biosystems, Foster City, Calif.).

GA1--GATCCATCGATGCGTCTGAATTAACACCAGCCGTGACAACTTACAAA

GA2--CTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACT

GA3--GAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAA

GA4--TACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGAT

GA5--GCGACTAAGACCTTTACAGTTACTGAAAAACCAGAAGTGATCGATGGATCC

GA6--TGTTAATTCAGACGCATCGAT

GA7--TGTTTTACCATTAATAACAAGTTTGTAAGTTGTCACGGCTGG

GA8--AGTAGCAGCATCAACAGCTTCAGTAGTTGTTTCGCCTTTCAA

GA9--GTCAACACCGTTGTCGTTAGCGTATTGTTTGAAGACTTTTTCTGC

GA10--AACTGTAAAGGTCTTAGTCGCATCGTCGTAAGTCCATTCACC

GA11--GGATCGGGATCCATCGATCACTTCTGGTTTTTCAGT

These polynucleotides are designed to hybridize to each other to formthe double stranded DNA sequence shown in FIG. 13.

Polynucleotides GA2 through GA10 are kinased by adding 1 ul of a 100ug/ml solution of each of the polynucleotides, 20 units of T4polynucleotide kinase to a solution containing 70 MM Tris-HCl at pH 7.6,10 mm MgCl₂, 5 mM DTT, 10 mM 2ME, and 500 ug/ml of BSA. The solution ismaintained at 37° C. for 30 minutes and the reaction stopped bymaintaining the solution at 65° C. for 10 minutes. The 2 endpolynucleotides, 20 ng of polynucleotide GA1 and 20 ng of polypeptideGA11 are added to the above kinasing reaction solution together with1/10 volume of a solution containing 20 MM Tris-HCl at pH 7.4, 2 MMMgCl₂ and 15 mM NaCl. This solution is heated to 70° C. for 5 minutesand allowed to cool to room temperature, approximately 25° C., over 1.5hours in a 500 ml beaker of water. During this time all thepolynucleotides anneal to form the double stranded synthetic DNA insertshown in FIG. 10. The individual polynucleotides are covalently linkedto each other to stabilize the synthetic DNA insert by adding 40 ul ofthe above reaction to a solution containing 50 MM Tris-HCl at pH 7.5, 7MM MgCl₂, 1 mM DTT, 1 mm ATP and 10 units of T4 DNA ligase. Thissolution is maintained at 37° C. for 30 minutes and then the T4 DNAligase inactivated by maintaining the solution at 65° C. for 10 minutes.The end polynucleotides are kinased by mixing 52 ul of the abovereaction, 4 ul of a solution containing 10 MM ATP and 5 units of T4polynucleotide kinase. The solution is maintained at 37° C. for 30minutes and then the T4 polynucleotide kinase inactivated by maintainingthe solution at 65° C. for 10 minutes.

The completed DNA insert is ligated directly into the pRIT5 protein Agene fusion vector shown in FIG. 11 that has been previously digestedwith the restriction endonuclease Bam HI. The ligation mixture is thentransformed into suitable host cells and DNA is prepared from theresulting single colony transformants. The sequence of the synthetic DNAinsert coding for the Protein G F_(C) -binding portion is determinedusing methods similar to or dependent on the chain terminationsequencing methodology described by Sanger et al., Proc. Natl. Acad.Sci., USA, 74:5463-5467 (1973). This DNA sequencing confirms theaccuracy of the above construction steps.

The synthetic DNA insert constructed above (FIG. 13), when inserted intothe Bam HI site of pRIT5 vector renders this Bam HI site uncuttable withBam HI. The synthetic DNA insert however contains a new Bam HI site. Thesynthetic DNA insert prepared in Example 9 containing the V_(H) -bindingpeptide p21-49 is inserted into this new Bam HI site. This is done bydigesting the vector constructed above with the restriction endonucleaseBam HI and ligating the synthetic DNA insert constructed in Example 9into this vector. The ligation mixture is then transformed into suitablehost cells and DNA prepared from the resulting single colonytransformants. The DNA sequence of the entire synthetic DNA insertcoding for both the Protein G F_(C) -binding portion and the V_(H)-binding peptide p21-49 is determined using the chain termination DNAsequencing methodology. This second round of DNA sequencing confirms theoverall accuracy of the series of construction steps performed togenerate a vector capable of expressing the Protein G F_(C) -bindingportion together with both the Protein A F_(C) -binding portion and theV_(H) -binding peptide p21-49. The Protein A F_(C) -bindingregion:Protein G F_(C) -binding region:V_(H) -binding peptide p21-49fusion protein is expressed in either E. coli or S. aureus using methodssimilar to the methods described in Nilsson et al., EMBO J., 4:1075-1080(1985); and Uhlen et al., Gene, 23:369-378 (1983).

The level of expression of the Protein A F_(C) -binding region:Protein GF_(C) -binding region:V_(H) -binding peptide p21-49 fusion protein isdetermined using an ELISA assay capable of detecting the V_(H) -bindingpeptide p21-49 in host cell extracts and on host cell membranes.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limitating. Numerous other variations andmodifications can be effected without departing from the true spirit andscope of the invention.

What is claimed is;
 1. A method of separating immunoglobulin moleculesfrom an aqueous sample, which method comprises the steps of:(a) admixingsaid aqueous sample with a polypeptide comprising a single polypeptidechain having a V_(H) -binding segment, selected from the groupconsisting of:(A) NH₂-Lys-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH,(B) NH₂-Lys-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lye-Ile-Leu-Gly-Asn-Gln-Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-COOH,(C) NH₂-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH,(D) NH₂-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lye-Ile-Leu-Gly-COOH,(E) NH₂-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH, and(F) NH₂-Lys-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-COOH,(b) maintaining said admixture under Ig-binding conditions to form abinding reaction admixture including a temperature of about 4° C. toabout 45° C., a pH value of about 5 to about 9 and an ionic strength ofno more than that equivalent to about one molar of sodium chloride for atime period sufficient for a portion of said immunoglobulin moleculespresent to bind said polypeptide to form a complex and a non-boundsample portion; and(c) segregating said complex from said non-boundsample portion, thereby separating said immunoglobulin molecules fromsaid aqueous sample.
 2. The method of claim 1 wherein said non-boundsample portion is recovered.
 3. A method of separating immunoglobulinmolecules from an aqueous sample, which method comprises the stepsof:(a) admixing said aqueous sample with a polypeptide that exhibitsantigen-independent affinity for a V_(H) region of antibody molecule,said polypeptide being free from a binding site for a humanimmunodeficiency virus, said polypeptide having an amino acid sequencerepresented by at least one of the formulae selected from the groupconsisting of: (A) NH₂-Lys-Lys-Ser-Ill-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH,(B) NH₂-Lys-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lye-Ile-Leu-Gly-Asn-Gln-Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-COOH,(C) NH₂-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH,(D) NH₂-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lye-Ile-Leu-Gly-COOH,(E) NH₂-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-Gly-COOH, and(F) NH₂-Lys-Lys-Ser-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-COOH,to form a binding reaction mixture; (b) maintaining said admixture underIg-binding conditions that maintain the biological activity of thepolypeptide and the antibodies sought to be bound including atemperature of about 4° C. to about 45° C., a pH value of about 5 toabout 9 and an ionic strength of no more than that equivalent to aboutone molar of sodium chloride for a time period sufficient for a portionof said immunoglobulin molecules present to bind said polypeptide toform a complex and a non-bound sample portion; and (c) segregating saidcomplex from said non-bound sample portion, thereby separating saidimmunoglobulin molecules from said aqueous sample.
 4. The method ofclaim 3 wherein said non-bound sample portion is recovered.